The rod-shaped, spore-forming, Gram-negative, anaerobe bacterium Clostridium perfringens is able to produce at least 17 toxins, making C. perfringens one of the most pathogenic species in the Clostridium genus. Depending on its ability to produce the four typing toxins, namely α-, β-, ε-, and ι-toxin, C. perfringens strains are classified into one of five toxinotypes, referred to as types A-E (Petit et al. (1999) Trends Microbiol. vol. 7, 104-110).
In addition to the typing toxins, the bacterium is able to produce a variety of so called minor toxins such as β1, β2, δ, θ, λ, μ, ν, and enterotoxin (Rood (1998) Annu. Rev. Microbiol. vol. 52, 333-360). Epsilon toxin (Etx) is produced by toxinotypes B and D. These strains are responsible for a severe disease called enterotoxemia, which affects predominantly sheep and lambs but also causes infections in other ruminant species, including goats and calves (Songer (1996) Clin. Microbiol. Rev. vol. 9, 216-234). Enterotoxemia in naturally infected animals is usually characterised by systemic lesions in sheep and enterocolitis in goats.
The most important factor in initiating disease is overeating rich food, resulting in the presence of high amounts of carbohydrates in the intestine. This leads to disruption of the microbial balance in the gut, leading to proliferation of C. perfringens and consequent overproduction of Etx. The toxin causes an increase in intestinal permeability, facilitating its entry into the bloodstream and allowing its dissemination to the main target organs of the kidneys and the brain (McDonel (1980) Pharmacol Ther 10(3): 617-655). Here, intoxication results in fluid accumulation due to increased permeability of blood vessels. Accumulation in the central nervous system results in neurological disorder rapidly leading to death (Finnie (2003) Aust. Vet. J. vol. 81, 219-221).
Etx is expressed with a signal sequence that directs export of the prototoxin from the bacterium (McDonel (1986) in Pharmacology of bacterial toxins eds. Dorner & Drew, Pergamon Press, 477-517). In development of disease, the relatively inactive prototoxin is converted to the active toxin by proteolytic cleavage in the gut lumen, either by digestive proteases of the host, such as trypsin and chymotrypsin (Bhown & Habeerb (1977) Biochem. Biophys. Res. Commun. vol. 78, 889-896), or by C. perfringens λ-protease (Minami et al. (1997) Microbiol. Immun. vol. 41, 527-535). Proteolytic activation of Etx can also be achieved in vitro by controlled proteolysis (Hunter et al. (1992) Infect. Immun. vol. 60, 102-110). Depending on the protease, proteolytic cleavage results in the removal of 10-13 amino-terminal and 22-29 carboxy-terminal amino acids (Bhown & Habeerb (1977); Minami et al. (1997)). Maximal activation occurs when both N- and C-termini are cleaved (Worthington & Mulders (1977) Infect. Immun. vol. 18, 549-551).
The 3D structure of Etx has been determined (Cole et al. (2004) Nature Structural & Molecular Biology vol. 11, 797-798) and reveals a molecule composed mainly of β-sheets, which can be divided into three functional domains. Domain I at the N-terminus contains the suggested receptor interaction region. Domain II in the middle contains an amphipathic β-hairpin, which is predicted to play a role in membrane insertion. Domain III at the C-terminus contains the C-terminal peptide, which has to be removed for activation to occur.
Epsilon toxin is an aerolysin-like β-pore forming toxin (β-PFT), with the amphipathic β-hairpin loops inserting into the membrane to form β-barrel structures. The overall fold of Etx shows similarity to the structure of aerolysin from the Gram-negative bacterium Aeromonas hydrophila (Parler et al. (1994) Nature vol. 367, 292-295), to parasporin-2 (PS) from Bacillus thuringiensis (Akiba et al. (2009) J. Mol. Biol. vol. 386, 121-133) and to a pore-forming lectin, LSL, from Laetiporus sulphurous (Mancheno et al. (2005) J. Biol. Chem. vol. 280, 17251-17259). The structural similarities between these toxins are most striking in their two C-terminal domains. Their N-terminal domains show a greater structural variation, which is likely to account for their differences in target cell specificities and potencies (Bokori-Brown et al. (2011) FEBS J. vol. 278, 4589-4601).
In aerolysin, the two amino-terminal domains (Domains I-II) are thought to play a role in binding to cell surfaces with overlapping functions (MacKenzie et al. (1999) J. Biol. Chem. vol, 274, 22604-22609) and it has been suggested that domain I of Etx, which is equivalent to domain II of aerolysin, performs a similar function (Cole et al. (2004)), but this has yet to be demonstrated. Domain II of aerolysin contains the mannose 6-phosphate binding loops. However, the residues of domain II involved in mannose-6-phosphate binding in aerolysin are not conserved in domain I of Etx, suggesting that the structural variation in the N-terminal receptor binding domains of these toxins is likely to account for the differences between their target cell specificities.
Etx is unique among β-PFTs because it is highly potent and has high cell specificity. Because of its high potency, Etx is considered to be a potential biological weapon for international terrorism by the U.S. Government Centres for Disease Control and Prevention (Morbidity and Mortality Weekly Report (MMWR) Recommendations and Reports (2000) vol. 49, 1-14). The 50% lethal dose (LD50) of Etx in mice after intravenal injection is typically 100 ng/kg (Gill (1982) Microbiol. Rev. vol. 46, 86-94), making Etx the most potent clostridial toxin after botulinum neurotoxin. Etx also shows high cell specificity. Among the many cell lines tested, only four have been identified to be susceptible to the toxin. These include kidney cell lines of dog (MDCK (Knight et al. (1990) Biologicals vol. 18, 263-270)), mouse (mpkCCDc14 (Chassin et al. (2007) Am. J. Physiol. Renal Physiol. vol. 293, F927-937)) and human (G-402 (Shortt et al. (2000) Hum. Exp. Toxicol. vol. 19, 108-116) and ACHN (Ivie et al. (2011) PloS ONE vol. 6, e17787)) origin. Most in vitro studies on Etx have been carried out using the Madin-Darby Canine Kidney (MDCK) cell line, as this cell line is the most susceptible to the toxin (Payne et al. (1994) FEMS Microbiol. Lett. vol. 116, 161-167). The dose of Etx to kill 50% of MDCK cells (CT50) is reported to be as low as 15 ng/ml.
The binding of Etx to MDCK cells is associated with the formation of a stable, high molecular weight complex (Petit et al. (1997) J. Bacteriol. vol. 179, 6480-6487). Intoxicated cells undergo morphological changes that include swelling and membrane blebbing before cell death (Petit et al. (1997) J. Bacteriol. vol. 179, 6480-6487). The rapid toxin-induced cell death and the specificity of epsilon toxin for only a few cell lines suggest the presence of a specific receptor(s) on target cells. The mechanism of Etx binding to cells is not known, but chemical modification studies of Etx have previously indicated that a tyrosine residue is necessary for binding of the toxin to target cells (Sakurai & Nagahama (1987) Toxicon vol. 25, 279-284). Toxicity appears to be a consequence of the formation of pores in the target cell membrane (Petit et al. (2001) J. Biol. Chem. vol. 276, 15736-15740).
Since progression of enterotoxemia from onset to death can be rapid in agricultural animals and given the potential for Etx to be used as a biological weapon, there is a need to identify molecules with potential for use as a vaccine against disease caused by or associated with the presence of Etx and/or caused by infection by C. perfringens. 